...net negative charge with 4 negatively charged and 1 positively charged amino acids. Myc tags are frequently...(stay tuned for our affinity purification tag blog!).   Applications of epitope tags   The...success rates across all applications. You can find FLAG antibodies on Addgene’s catalog including a mouse monoclonal...protein purification is not ideal with Myc, as low pH conditions are required during the purification, which...experienced. Finding an antibody that works for your application, specifically detects your protein, is species...and have been widely used across species and applications. In this blog, we will review the most common...that are bound by antibodies for the purpose of purification or detection of the tagged protein. These peptides...

...into three main effector functions.  Figure 1: Fc effector functions in an immune response to a ...determination, don’t generally affect antibody applications like ELISAs or Western blots, they are key in...

...motion videos is very time consuming; producing a 1-2 minute video takes an entire week of our free time...Research 5 Reasons to Use Reddit for Science Communication 10 Ways to Share Your Science Resources on ...

...the brain where DA quantitation is desired (Figure 1). The probe perfuses liquid, which causes the passive...at a certain frequency, and this frequency can indicate behavioral state, for example, if someone is awake...

... Table 1: New ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor...

...materials to the research community. References 1. Coroadinha, A. S., et al. "Effect of osmotic pressure...AAV yields. Given the wide range of downstream applications scientists rely on our AAV preparations for,...

...enables the high-resolution tracking of more than 1 million cancer cells under drug treatment.” Stegmeier... interference. In the first two months since publication, the lab got over 100 requests for the plasmids...

...References 1. Huang, Ally, et al. "BioBits™ Explorer: A modular synthetic biology education kit." Science...plasmids and their potential educational uses. We believe that biology education is something that should ... expensive tiny amounts of reagents are, how complicated (and again, expensive) the different machines...desire to create a set of low-cost, easy-to-use educational biology activities – that any school could afford...proteins without the need to handle a living, replicating organism. Furthermore, cell free systems can ...in adapting your research ideas into hands-on educational activities using the BioBits platform, please...BioBits™ Bright: A fluorescent synthetic biology education kit." Science Advances 4.8 (2018): eaat5107. PubMed...

...difficult to modify in the past.   Figure 1: Identification and enrichment use of XMAS-TREE. a) Cells ...  After identification of ABE activity, XMAS-TREE can also be used to purify these modifications and work...ABE-targeted cells By Ellie Adams Single base modifications through ABE or CBE's have the potential ability...point mutations. They allow single-nucleotide modifications to edit DNA without requiring a double-stranded...mCherry and GFP expression to check for ABE’s. b) Quantification of base-editing efficiency using XMAS-TREE-...methods at various genomic loci in four different categories.     Brookhouser, N., et al. BMC Biol 18...MYC-dependent cancer cell lines. It can be applied for identification of essential MYC binding sites in a wide range...

...vesicles. Okada CY, Rechsteiner M. Cell. 1982 May;29(1):33-41. PubMed. Protein transduction: unrestricted...protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only...the HIV protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may ...that components in the buffer used for protein purification must be helping the untagged protein enter cells...Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP using Cre-lox recombination...showed up to 70% resistance to diphtheria toxin, indicating a 70% frequency of biallelic mutations. Importantly...Importantly, hESCs retained their stemness, indicating that iTOP may be valuable for CRISPR/Cas9 stem cell...

...occur, but good performers move around easily  Table 1: Taking it a Step Further - Big Company v. Small Company...scientific and technical training is that these educational paths prepare you for a vast selection of career...tech transfer, management consulting, science communication, policy and the diverse options in nonprofit...science is much like industry but the “product” is publications—the pressure to produce is the same You get ...and teach students Collaborations may be less “complicated” - for example, easy to initiate a collaboration...is different about non-academia? Scientific publications are a nice to have, not a need to have Must ...every 3-6 years—a short and exciting ride Complicated matrix structures are common Small integrated...

...Article contributed by Susanna Bachle Figure 1: Initial steps in the CRISPR-pooled screening using...infection, because this setup allows for experimental replicates for each of the gRNAs. The idea was to assign...cassette is removed, the effector is expressed. By truncating one of the recombination sites in the construct...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ... reporter (blue) and stained with PI (pink) to indicate cell death. Image from Diessl et al., 2020. ...https://dx.doi.org/10.15698%2Fmic2020.04.713 Truncated TALE-fluorescent protein fusions as a DNA staining...protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison...

...do researchers track fluorescently labeled cells? 1. Epifluorescence microscopy This technique uses a ...of the immune system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals... required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...relatively little tissue absorption, weak tissue scattering and small amounts of tissue auto-fluorescence...

... contain the full length plasmid.   Figure 1: The full lentiviral plasmid is expected to be >12... (AmpR) and origins of replication (ori and f1 ori) which allow it to replicate.   How can I prevent...the bacterial selection marker and origin of replication. During bacterial culture, only the plasmid with...the antibiotic resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria...the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the full...

...extension. Now, they split EB1 into two halves (Fig. 1): one containing the microtubule binding domain and...cells, and has potential use for a wide range of applications in cellular biology. Van Haren J et al. Nature...versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9 cleavage activity ...plasmid that allows for rapid fluorescence-based quantification of base editors that utilize dCas9 fused to... The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry fluorescence is...frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply involves tracking...

...variants. This resulted in the construction of redCALWY-1, a red-shifted FRET sensor for zinc using variants...Neurobiology has been working on genetically encoded indicators of calcium and other small molecules since the...lymphocytes, too, making them a versatile tool for application in both neuroscience and immunology. Don’t be...imaging for beginners Griesbeck says his calcium indicators can be used in mouse, Drosophila, C. elegans,...give it a shot. “Beginners sometimes think it’s complicated, but actually it is very good,” he said. “It ...his team is now applying it to improve upon an indicator they developed and published in 2010 for visualizing...

...heat-fixed smear with the crystal violet stain for 1 minute. Rinse the slide briefly with tap water. Flood...ll be talking about the ins and outs of this classification system, including its history, biology, and...repeatable phenomenon, Gram began using it as a classification system. Eventually, any bacteria that retained...permeable to different classes of antibiotics. The classification system is also useful in a laboratory setting...bacteria type. The gram positive and negative classification system is incredibly helpful in visualizing...stepping into a lab.   Anna Kamens is a science communication summer intern at Addgene and a rising third-...

... cell types and physiological states. References 1. Ingolia, Nicholas T., et al. “Genome-wide analysis... While ribosome profiling co-opts the physical location of the ribosome, TRICK (Translating RNA Imaging...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...ribosomes after initiation, to calculate a ribosome translocation rate of ~3.5 codons/second. They also explored...Resources the Addgene Blog Learn More about SunTag Catch up on Your GFP Background Choose the Brightest Fluorescent...

...mature HA-tagged proteins in vivo. Nature Communications, 10(1). PubMed PMID: 31270320. PubMed Central ...antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based...

...Transcriptional Programs with CRISPR RNA Scaffolds. Cell, 160(1), 339-350 (2015). PubMed. Find the Plasmids @Addgene... interference (CRISPRi) model consists of a catalytically dead Cas9 protein (dCas9) that lacks endonuclease...library of characterized aptamers and supporting publication: http://www.freebase.com/base/aptamer Cruz-...

...process is explained more fully in the animated Fig. 1 below. Normalization is a fundamental and often understated...smaller fragments that are then barcoded for identification. This mixture of barcoded fragments is known...of Toronto. He is an author of 20+ scientific publications and patents.   ...

...heterodimerizes with CIBN upon blue light exposure (see Fig 1*). When the group targeted the enhancer region of ...plasmids at Addgene Opperman J et al., Nature Communications, 2019. PubMed PMID: 31346176. Using LADL to...light-activated-dynamic-looping (LADL) system. The group fused dCas9 to a truncated CIB1 protein from Arabidopsis (CIBN), which they...

...post about multiplexing with Cpf1.   Figure 1: Multiplexing allows researchers to express multiple...be used in a wide range of bacteria. References 1. Maddalo, Danilo, et al. “In vivo engineering of oncogenic.... Using multiplexed gRNAs can target multiple locations in the genome to modify, whether it be editing... use is pX333 from the Ventura lab. pX333, a modification of pX330, contains humanized wtCas9 and two ...thus marking cells that have undergone genomic modification. In this system, Cas9 must be supplied on a ...with Cpf1 Additional Resources on Addgene.org Catch Up on Your CRISPR Background with Our Guide Pages...

...present in the data such as synapsin, hsyn synapsin-1, human synapsin, hSynap. Matching We capture multiple...that allows category-specific, narrow searches. We defined three top level categories — catalog, educational... in a given category, and serve as a shortcut to a search scoped to that category. Catalog ID suggestions...prefer to browse and discover items in our catalog or educational resources through our main dropdown menu... of Addgene’s customer’s needs. Categories We created a categorization of our information in order to ...depositors — each with subcategories such as plasmids and viral preps for catalog, blog posts and protocols...80,000+ reagents and 700+ educational resources — including blog posts, protocols, guides, and collections...

...red light rapidly deactivates the system. Version 1 of this far-red/red light-responsive system is spread...consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength can... also be used to engineer new pathways with sophisticated gene expression controls. Most cell types do...light; these two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive...; only two plasmids are required per system. Applications of these systems The large dynamic range of ...suitable for various scientific or engineering applications.   References Refactoring and optimization ...Oct 11. PubMed. Find the plasmids from this publication at Addgene. Synthetic biology: engineering Escherichia...

...instead conveniently provide them on your smart phone. 1. Lab Archives by LabArchives The single, most important...Gibco organizes its app into four categories: tools, analytics, education and data manager. The tools are...are five FREE science lab apps that have been educational and great for lab life. These apps prevent you...lab notebook. Lab Archives is a multipurpose application that allows you to share your data with colleagues...other nice features. This app also has a great educational section. This section holds the Gibco Cell culture...directions including which buffers to use and that indicates if a sequential digest is needed. The enzyme finder... to this app; the user can search by sequence, category of enzyme, or name to discover all the information...

...intern to complete and present on.    Figure 1: Addgene's summer interns presenting their capstone...compiled a list of three options from the Addgene catalog. These were a miR30 cassette, Syntaxin3, and osa-miR528...

...to do that than in the Addgene blog?  Fig. 1: One of our favorite content collections is our Help...Our educational content  Have you ever wondered how we choose topics for the Addgene blog? Or why we...everything else we do! The goal of Addgene’s educational content Addgene truly believes that sharing speeds...antibody and content team, we identified what applications should be included and what basic information... The guide was then assigned to our science communication specialist, Rachel, to write. Identify expertise...through an editing process and final check before publication.   Do research! Nearly everything we write entails...Guide figures. This requires a fair amount of communication between our content team and our graphic designer...

...and cost for AAV genome sequencing.  Figure 1: The viral genome sequencing workflow. Using this...that could be used as a template for downstream applications (Berns, 1972). Using qPCR and restriction enzyme...

... After all I was only going to be interviewed for 1 hour so having a broad enough insight was going to...there a move with either promotions or further education may allow you to transition into various realms...

...defunct (but fun) plug-in called Maps.  Figure 1: See a starfish? This is actually a map of Joanne ...FiercePharma), or sign up to be alerted about their new publications via Pubmed, so you can congratulate them when...

...participants could share their notes together. Figure 1: Can you draw Spongebob from these instructions without...session to another,” Bachle says. “Now, the physical location didn’t matter. I didn’t have to consider that ...

...faster. Years of science, done in weeks Figure 1: Sending out 80 FedEx envelopes in a single day --...including more physiologically-relevant systems, to catch all the important interactions. It’s also important... we found in our map have an impact on virus replication. While we and our collaborators are starting ... Nsp3 and Nsp16, and includes an additional catalytically dead version of the viral protease, Nsp5. Our... 2xStrep-tag for easy detection and affinity purification. All but one are in a pLVX vector background...coronavirus, one FedEx package at a time Since the publication of our paper, our group has been inundated with...sending plasmids to all who request them -- in locations all over the world, as far flung as the EU, Russia...

...-viral applications are also possible through the use of nanoparticles. [6].    References 1. Zhang ...2020.  “CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future.” Front. Oncol...with a dominant mutation in a gene that causes cataracts could be rescued by coinjection into zygotes of...the technology in different labs for different applications.” Li’s team noted that CRISPR/Cas9 has been ... explore its potential in mice with a dominant cataract disorder caused by a single copy of a gene known....  “CRISPR Genome Editing Technology and its Application in Genetic Diseases: A Review.” Current pharmaceutical...

...into account, the digest pattern depicted in Lane 1 would be expected for a XbaI/NotI digest. The program... one half of naturally occurring restriction modification systems that prokaryotes use to protect themselves... has genes encoding one or a few restriction modification systems containing methyltransferases that add... phage for instance). Aside from restriction modification systems, DNA methylation also plays an integral...integral role in regulating genome replication, repairing mismatched basepairs or small indels that occur...methylases) are independent from the restriction modification systems, yet can still affect whether certain...specifically associated with any restriction modification systems, their sequences may overlap with restriction...

...vector-exosomes), exosome AAVs, or exo-AAVs (Figure 1). The researchers discovered that these particles ...the classical purification protocol involving iodixanol gradients. The current purification protocols for...particles accumulate inside the producer cells and purification protocols often describe a process that begins...lysis to release AAV particles with subsequent purification steps. It’s also possible for AAV vectors to... This is particularly useful for therapeutic applications in vivo where endogenous anti-AAV antibodies...Adrienn, et al. "Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous...

...pitfalls? Here's what you need to know.   Figure 1: Management can seem a mystery at first! Image used...cohesive team environment. Neglecting communication skillsClear communication is the backbone of effective management...management. Miscommunication often arises when managers fail to set expectations or provide timely feedback...firsthand when it took me some time to adapt to the communication style of a new, junior team member who had values...and leveraging tools like the Myers-Briggs Type Indicator or DISC assessments can help you navigate team...

...as an endogenous RNA imaging tool.   Fig. 1: Csm, shRNA, and Cas13 were compared head-to-head ...antibodies may just not perform as well in one application as another, so it is always best to have options...for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page for more...fluorescent proteins (FPs) necessary for many of these applications have lagged. Fixation, chemical staining, and...well-suited for the demands of advanced microscopy applications such as super resolution, electron, and expansion...

...the guides are relatively unstable.   Figure 1: CRISPRainbow labeling. Two MS2, PP7, or boxB sequences...The catalytically dead Cas9 protein (dCas9) is well known for its ability to bind DNA targets without...has been widely adapted for a wide variety of applications: base editing, CRISPR activation and inhibition... (B) CRISPR-Sirius used to visualize relative locations of the subtelomeric region of the pericentromeric...al. “CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging.” Nature Methods 15 (2018)...

... genes with a single viral vector.   Fig. 1: Schematic of multiplex gene editing with DAP array...packaged into viral vectors for potential in vivo applications or multiplex CRISPR screening.   Using tRNAs...multiplex genome engineering of crops or allow for sophisticated CRISPR screenings that would perturb the unprecedented...and efficient in plasmid and viral vectors. In applications when repetitive tRNA sequences must be avoided...construct DAP array for multiplex gene editing applications, we provide detailed manual and online design...

...databases, EENdb (published 1 Jan 2013, no longer available), is a simple catalogue of reported sgRNA designs...follow him on Twitter @CMacPhD.          References  1. Ghorbal, Mehdi, et al. "Genome editing in the human...into this category describe how the user is expected to interact with the software. This category is important... CRISPR software submissions. With few prior publications to go on, a submitted manuscript could only ... solutions that facilitate most experimental applications in CRISPR engineering. When compared to that...easy to fault the lack of features in a single application. The question is then, should we use all tools...novo sgRNA design, I again separate it into two categories. On the one hand we have software tools that ...

...settings with experimental samples. Figure 1: Example flow plots for a single color control. A)...ll need a few controls specific to flow as an application. These controls will allow you to distinguish...commercial antibody pages. Flow cytometry can be a complicated and daunting process. Controls like single color...

...and also by a lactose analogue, IPTG (isopropyl β-D-1-thiogalactopyranoside). (To be completely accurate...insert. All colonies on this plate should be blue, indicating that your IPTG and x-gal are working as they ...of false positives: Blue-white screening only indicates the presence of AN insert, not necessarily YOUR...

...Networking 101-Building Relationships for Success”. Choose 1-2 tactics to try. One of my favorites is making the...great way to get started with mentoring. Read this Catalyst Report “Making Mentoring Work” to learn about ...tapping into it and helping make it work by being an educated mentee. Other opportunities: Try virtual mentoring...

...medRxiv.org) in the past few years.   Fig. 1: Stand aside, CERN - it's a new age of accelerating...their CV or grant applications. A number of organizations (such as ASAPbio) advocate for preprints, and...the most important part of your research is communicating the results of the work. Yes, those control ...any impact! There are lots of ways scientists communicate with each other: from formal mechanisms like ...poster or talk, or submitting patent or grant applications; to informal settings like lab meetings, Twitter...of the most significant shifts in scientific communication is the rising use of preprints, or scientific...publishing in a journal.  Accelerating Scientific Communication Preprints have been common in fields like math...

...antibody that doesn't work for your application.      Fig. 1: Antibody Selection Flow Chart  ...your specific application. Consider also that there may be variations within an application. Take immunohistochemistry...website highlights a variation of your application, a publication successfully used it in a similar way...choosing an appropriate antibody first look at the application-specific data provided on the manufacturer’s ... a linear antigen tend to work very well for applications that denature a protein to its primary (linear...the most similar to yours. Take a look at the publications cited on the manufacturer’s website, which may...plan to. To secondary or not to secondary?  The application will also determine whether you should use an...

...techniques?  Let us know in the comments! References 1. Bacteriophage P1 site-specific recombination. I. ...recombinase catalyzes the site specific recombination event between two loxP sites, which can be located either...maintained).  Translocation: If the sites are on separate DNA molecules, a translocation event is generated...important plasmid elements – promoters, origins of replication, protein tags, and antibiotic resistance markers...creating (excuse the pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells,...proteins or co-factors, thus allowing for broad applications in a variety of experiments. LoxP (locus of ...and the canonical loxP sequence is ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT. The loxP sequence does not occur...

... contact her at [email protected]. References 1. Dieterich, Daniela C., et al. "In situ visualization...This application of AHA labeling is known as fluorescence noncanonical amino acid tagging (FUNCAT, Dieterich...experiments. Visualizing newly synthesized proteins with FUNCAT Professor Schuman’s technique applies metabolic...the whole de novo synthesized proteome. However, FUNCAT alone cannot be used to determine the turnover ...specific protein. Professor Schuman’s lab showed that FUNCAT’s usefulness can be extended to determine the turnover...ligation assay (PLA). The clever combination of FUNCAT and PLA PLA was originally published by the Söderberg...DNA strands take part in rolling circle DNA amplification. The amplified DNA segment is labeled with fluorescent...

...efficiently with adenovirus and AAV.   Table 1: Types of viruses and their features Choosing ...injections, or the location of a specific cell type doesn’t always lend itself to direct application of virus. ...addition of a helper virus or plasmid allows it to replicate episomally, essentially remaining an extrachromosomal...extrachromosomal part of the host genome that can replicate and divide along with the endogenous DNA. AAV also...gamma-retro viruses. This is large enough for many applications, but in some cases multiple AAVs (co-infection...and AAV. If you’re wondering more about what applications these viruses are good for, we have plenty of...Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New...

...guide outlines these components.     Fig. 1 - Viral vector components of retrovirus  Adeno-associated...ITRs serve as a type of origin of replication to initiate replication of the dsDNA virus. Viral packaging...plasmid while the other plasmids with packaging or replication components are called helper plasmids. Below,... production, they can be helpful for certain applications and aid in ease of use. Our lentiviral guide...AAV are rep and cap. Rep is required for viral replication and also aids in infrequent genome integration...a helper plasmid and mediate efficient viral replication. More information on these viral genes is available...particles. E2A/E2B are responsible for viral replication, with E2B being the primary viral polymerase....